By virtue of recent advancement of immunochemistry, immunoassays which ensure sensitive detection of a trace amount of a substance by use of an antigen-antibody reaction are widely used. A common immunoassay is, for example, immunostaining methods.
Immunostaining methods are intended to detect a specific substance on a cell or a tissue section using, for example, an antibody which recognizes the substance. Among these methods, those using an enzyme as a detectable substance are referred to as immunoenzyme techniques. Methods which have been developed as the immunoenzyme techniques include direct methods using a primary antibody labeled with an enzyme capable of visualizing the antigen and indirect methods comprising labeling a secondary antibody without labeling a primary antibody.
Further, in recent years, further high sensitivity is required in order to visualize a small amount of an antigen protein distributed in tissues and cells or to verify an antigen substance whose antigenicity is significantly impaired by formalin fixation or paraffin embedding treatment, and various amplification methods, which are modified immunoenzyme techniques, have been developed one after another. Examples of those amplification methods include, in ascending order of sensitivity, direct method<indirect method<PAP (peroxidase anti-peroxidase) method<ABC (avidin-biotin-peroxidase complex) method<LSAB (labeled streptavidin biotin) method<polymer method<CSA (catalyzed signal amplification) method.
(Non-Patent Document 1)
Among the above amplification methods, the most popular, highly-sensitive and simple methods are, at present, polymer methods.
One conventional polymer method is, for example, a method comprising reacting a primary antibody with a target marker such as an antigen, and then reacting a polymer reagent (in which many enzymes and secondary antibodies are bound to a polymer) with the reaction product, thereby forming a complex of the antigen, primary antibody, secondary antibody, polymer and enzyme. Color development of a substrate through the use of the enzyme activity in this complex allows visualization of the target marker (Non-Patent Document 1).
Also, another polymer method is, for example, a method comprising reacting a bridge reagent (which is also named variously by makers, e.g., linker, probe or post-primary) between the primary antibody and the polymer reagent in the above conventional polymer method for signal amplification. It is said that, as a result, this method can be expected to have twice to five times as high sensitivity as that of the above method (Non-Patent Document 1).
In recent years, there has been further developed a new polymer method comprising reacting an additional polymer reagent (second polymer reagent) with the above polymer reagent (first polymer reagent) for signal amplification (Patent Document 1).
However, even when the conventional polymer method is employed, staining at a desired level cannot be attained, in some cases, because of an extremely small amount of the target marker, decreased antigenicity, and so forth. In such a technical situation, a means for detecting a target marker simply and with higher sensitivity is still demanded.